Upload
others
View
10
Download
0
Embed Size (px)
Citation preview
Table S1. ESTs of putative bag genes from various plants (obtained from the EST
database search using default parameters of NCBI BLAST).
Plant
name
Library description EST
Accession number
Amino acid
similarity to BD of
AtBag4 (%)
ABIOTIC barley
(Hordeum
vulgare)
sorghum
(Sorghum
bicolor)
soybean
(Glycine max)
sugar beet (Beta
vulgaris)
grape (Vitis
vinifera)
BIOTIC barley
(Hordeum
vulgare)
grape (Vitis
vinifera)
legume
(Medicago
truncatula)
TISSUE - hot pepper
(Capsicum
annuum)
legume
(Medicago
truncatula)
sorghum
(Sorghum
bicolor)
grape (Vitis
vinifera)
STRESS seedling shoot (2 days of cold
stress)
mix of 5-week old plants (7 and
8 days of water stress)
3 days drought stressed leaf
tissue of one month old plants
4-day germinated seedlings
under stress (salt/NaCl,
dehydration/Mannitol and
anaerobic stress) mixed 8, 9, 11, 13, 15, 16 weeks
abiotic stressed berries
STRESS seedling green leaf (Blumeria
challenged)
mid-season development leaves
infected with the bacterial
pathogen Xylella fastidiosa
elicited root tissues derived cell
culture (induced with yeast cell
wall extracts)
SPECIFIC EXPRESSION flower bud
developing flower
ovary
developing preanthesis pannicles
berries (post-veraison stage)
fruit (green stage)
BF622329
BF622327
BE592352
BE593047
BG043600
BI073238
CB912188
BE216889
CB342561
BF646049
BM066508
BI272171
BG356714
BI140027
CB980485
CB980084
CB980018
BQ799356 BQ799253
60
62
67
67
59
43
77
62
54
59
73
62
62
48
79
79
79
66
65
cotton bolls
(Gossypium
arboreum)
potato (Solanum
tuberosum)
wheat (Triticum
aestivum)
soybean
(Glycine max)
apple (Malus x
domestica)
pine (Pinus
pinaster)
berries (veraison stage)
fibers isolated from bolls
mature tuber
roots
roots of five day old etiolated
seedlings
seedling shoot
immature seed coats
cultured shoots
differentiating xylem
BQ798817
BQ798556
BQ798294
CB347936
BQ403711
BF268630
BF460358
BM108950
BE405084
BE426477
AI960691
AU223481
BX249429
65
65
54
65
44
42
56
58
64
62
59
63
61
Methods
In Vitro Protein Binding Assays
The plasmid pGEX2T-701, a GST fusion of the Arabidopsis hsc70-1 gene (accession
number – X74604), was kindly provided by C. Guy, University of Florida. GST-Hsc70
was expressed in E. coli BL21 cells as described (Takayama et al., 1995) and purified
using Glutathione Sepharose 4B in accordance with the Amersham Health Inc. protocol.
A GST-Bcl-2 fusion protein was obtained from T. Ostersdolf (Idun Pharmaceuticals,
Inc.).
To determine whether or not the BAG domain of AtBAG4 is responsible for binding to
Hsc70, site-directed mutagenesis of this domain was performed. Two binding residues
were substituted using following primer pairs: 5’-
CCGCTCGAGCGGCATTGTCGAAAAACGATGATGC-3’ + 5’-
TCATAAGAAGCCCTGCAGCCATG-3’ and 5’-CATGGCTGCAGGGCTTCTTATGA-
3’ + 5’-GCTCTAGAGCATGTCAGTCAAATTTCTCCCAATC-3’ (AtBAG4m1:
Glu179 (Fig. 2B) (corresponding residue of hBAG1 E212, Fig. 2A) ! Gly; A XhoI site
was introduced at the 5’ end and a XbaI site at 3’ end ). AtBAG4m1 was cloned into
TOPO vector and amplified in E coli (Invitrogen). Isolated plasmids were used as
template for introduction of the second point mutation. The second point mutation was
introduced with following primer pairs: 5’-
GGTGAATTCATGATGCATAATTCAACCGAA-3’ + 5’-
ATTCCGGACAATTTGAGCAGCTGCCTCATA-3’ and 5’-
TTGTCCGGAATTGAGGCTGAAGGGGACG-3’ + 5’-
CCGCTCGAGTCAGTCAAATTTCTCCCAATC-3’ (AtBAG4m1m2: Asp189 (Fig. 2B)
(corresponding residue in hBAG1 D222, Fig. 2A) ! Ser by introducing a BspE1 site;
EcoR1 at 5’ and XhoI at 3’ end). The deletion of the BAG domain was made using
following primer pairs: 5’-GGTGAATTCATGATGCATAATTCAACCGAA-3’ + 5’-
TCCCCCCCGGGCACCGCAACTTGCGTCCCTCCA-3’ and 5’-
TCCCCCCCGGGAACTTGCAGGAGGCTGTGGATA-3’ + 5’-
CCGCTCGAGTCAGTCAAATTTCTCCCAATC-3’ (BAG-del: all conserved amino
acid residues within the BAG domain removed (Fig. 2A) + XmaI site introduced; An
EcoR1 site introduced at 5’ end and a XhoI at 3’ end). AtBAG4m1m2 (BAG-mut) and
BAG-del PCR products were digested, ligated into pCDNA3(+) and cloned into E. coli.
In vitro translated, [35S] labeled AtBAG4, BAG-mut and BAG-del were synthesized from
pAtBAG7.1 (wt), BAG-mut in pCDNA3(+) and BAG-del in pCDNA3(+) using TnT
Quick Transcription/Translation System (Promega). 2 !g of pAtBAG7.1 plasmid DNA
was incubated with 40 µl of TnT Quick Master Mix containing 20 µCi of [35S]methionine
at 30°C for 90 min. The purified GST-proteins were immobilized with 10 µl of
Glutathione-Sepharose beads and incubated with 5 !l in vitro translated AtBAG4 in 275
µl of binding buffer (50 mM Tris-HCl, ph 7.6, 150 mM NaCl, 0.1% Triton X-100) at 4°C
for 3 h. The beads were washed three times with 1 ml of binding buffer, resuspended in 5
!l of SDS-loading buffer, boiled for 3 min., and subjected to SDS-PAGE, followed by
exposure to x-ray film. The use of equivalent amounts of GST-fusion proteins and in
vitro translated AtBAG4 was confirmed by SDS-PAGE analysis, followed by Coomassie
staining, or autoradiographically, respectively.
Plasmid Constructions and Plant Transformation
The following PCR primers: 5’–TGTCGAAAAATCATGATGCATAATTCAACCG –
3’ and 5’–CCAAGGCAACGGAGCTCTACTAAAATGTC–3’, were designed such that
a BspH1 site introduced at the 5’-end of the AtBAG4 cDNA and Sac-I site at the 3’-end.
PCR was performed with pAtBAG7 plasmid DNA. The PCR product was digested with
BspH1 and Sac-I and subcloned into the plant expression cassette pRTL2. The expression
cassette containing atbag4 under the control of the cauliflower mosaic virus 35S
promoter and the Agrobacterium nopaline synthase terminator was subcloned into the
binary vector pPZP211 as HindIII fragment. The resulting construct is referred to as
pZPRTL2BAG. The binary vector pZPRTL2BAG was mobilized into Agrobacterium
tumefaciens strain C58C1 by triparental mating for further tobacco transformation.
Agrobacterium transconjugants were selected on YEP medium supplemented with
rifampicin (50 mg/liter), gentamycin (50 mg/liter), spectinomycin (100 mg/liter), and
streptomycin (100 mg/liter). Tobacco leaf tissue (cv. Xanthii, genotype NN) was
transformed and plants were regenerated as described (Horsch et al., 1985).
Analysis of gene expression
Total RNA from different tissues (leaf, stem, flower, and root), leaves at different ages,
and at different time points after heat treatment or after cold stress, was extracted using
TRIzol reagent (Invitrogen). For first-strand cDNA synthesis, RT-PCR was performed
using the following components: reverse transcription buffer (50 mM Tris-HCl ph 8.3, 50
mM KCl, 10 mM MgCl2, 10 mM DTT, and 0.5 mM spermidine), 400 µM of each dNTP,
40 µM random primer, 2 µg total RNA, and DEPC-treated water added up to 25 µl final
reaction volume. AMV Reverse Transcriptase (10 units, FisherBioReagents) was added
and the reaction incubated at 55°C for 30 min. The resulting RT-PCR fragments were
amplified using specific primers designed from the coding region and spanned the
intron/exon junctions of atbag4 (5` - TTCTACGTACCTCAGC-CTTACGC - 3` and 5` -
ACGCCACTTTCGGGGATGTAAAG - 3) or atbag6 (5` -
TGCTGTCGAGGGATTGCATCC - 3` and universal primer 5` -
TCACTAGCATTCTCCGGGTCC - 3) to eliminate possible DNA contamination.
Expression from the Arabidopsis actin1 gene using specific primers also spanning
intron/exon junctions (5` - TCGTACTACCGGTATTGTGCTCGAC - 3` and 5` -
TTTGCGATCCACATCTGCTGGAAGG - 3`) were used to ensure equivalent RNA
loading.
Figure Legends
Figure S1. Gene structure of the Arabidopsis BAGs. Similar exon-intron organization
within the BAG were found in genes, the products of which are predicted to localize in
the same subcellular compartment (see text for details).
Figure S2. In vitro binding assay of AtBAG4 to Arabidopsis Hsc70. GST-Hsc70 was
immobilized on glutathione-Sepharose beads and tested for in vitro binding to in vitro
translated 35S-labeled AtBAG4. Samples were analyzed by autoradiography to detect
bound AtBAG4 to the immobilized Hsc70 protein (“pull-down”). Binding of AtBAG4 to
GST coupled to glutathione-Sepharose beads was used as an unspecific binding control.
In vitro translated 35S-labeled AtBAG4 was loaded in the gel to compare the amount of
input versus bound AtBAG4 (“supernatant”). BAG: AtBAG4, BAG-mut: AtBAG4 with
two mutated binding residues, BAG-del: AtBAG4 without BAG domain.
Figure S3. RT-PCR analysis of atbag4 and atbag6 expression patterns. A. The atbag4
and atbag6 transcript abundance in various tissues of flowering Arabidopsis plants.
Arabidopsis leaf tissue of prior to and during flowering plants was also included to
compare atbag4 and atbag6 expression in different developmental stages. B. atbag4 and
atbag6 transcripts levels are differentially regulated by cold treatment. Arabidopsis
flowering plants were cold treated at -20ºC for 10 min. and leaf tissue samples were
collected at 0 min., 20 min., and 90 min. after exposure. C. atbag4 and atbag6 transcripts
levels are differentially regulated by heat treatment. Arabidopsis plants were heat treated
and leaf tissue samples were collected at 0 min., 30 min., 60 min., and 90 min. of
exposure to 37ºC. Actin-1 expression was used as a control of total RNA equal loading to
RT-PCR reaction mixes.
Figure S4. Oxidative stress resistance in atbag4 low and intermediate level expressing
transgenic tobacco plants. Phenotypes of detached wild-type and selected transgenic (L-2
and I-5) tobacco leaves after two weeks of 5mM menadione treatment.
Figure S5. Phenotypes of UV-irradiated leaf discs from wild-type and transgenic
tobacco plants: L-2, I-5 and H-12 (atbag4 high level expressing line) one week after
treatment with UV-C light (32kj/m2).
Figure S6. Mean number of live leaf discs from wild-type and transgenic tobacco
plants L and I lines after 24 and 48 h of 10 !M paraquat treatment. Leaf discs were
scored as dead when their disc areas were stained more than 20 percent by Evans blue.
Figure S7. Freezing tolerance in low-level atbag4 expressing line. A. Chlorophyll a
and b contents in wild-type and transgenic leaves 24 h. after the cold stress at -20°" for
10 min. Chlorophyll was extracted with 80% aqueous acetone. The concentrations of
chlorophyll were determined spectrophotometrically at 652 nm. The content is expressed
as a percentage of the mock leaves. B. Mean number of live leaves after 40h cold stress
at -20°" for 6 min. Leaves were scored as dead when their leaf areas were stained more
than 20 percent by Evans blue.
Figure S8. DNA fragmentation in cold stressed wild-type and low level atbag4
expressing tobacco leaves 2h. after cold treatment at -20°" for 10 min. Ethidium
bromide-stained agarose gel of DNA isolated from the transgenic line L-17 (lane1) and
wild-type tobacco (lane 2 and 3) after cold stress, and L-17 (lane 4) and wild-type
tobacco (lane 5) without any treatment.
Figure S9. RT-PCR data of atbag4 and atbag6 expression patterns in response to (A) B.
cinerea stress, and (B) hormone treatment; in (C), atbag4, atbag5, and atbag6
expression pattern in response to divalent metal ions treatment are shown( lane5, the
plants were pretreated with EDTA 12 h. before Ca2+ treatment). Arabidopsis plants
without any treatment exposure were used as a reference control. Actin expression was
used as an equal loading control.
Supplementary fig. 1
Supplementary fig.2
Supplementary fig.3
Supplementary fig.4
Supplementary fig.5
Supplementary fig.6
Supplementary fig.7a
Supplementary fig.7b
Supplementary fig.8
Supplementary fig.9